| 摘要 |
A template-mediated, ligation-oriented method for randomly shuffling polynucleotides comprising adjacently hybridizing at least two fragments on an assembly template, is new. A template-mediated, ligation-oriented method (M1) for randomly shuffling polynucleotides comprises: (a) obtaining, directly or indirectly, from a polynucleotide library, single-stranded fragments of at least two homologous polynucleotides; (b) hybridizing the fragments to one or more devised assembly templates until at least two of the fragments are adjacently hybridized to form at least one partially double-stranded polynucleotide, where at least one of the templates shares at least one zone of homology with the homologous polynucleotide; (c) treating the partially double-stranded polynucleotide, in order to form at least one recombinant polynucleotide, by: (i) ligating nicks; and (ii) where necessary, any one of or any combination of the following: (1) filling in gaps by further hybridizing the fragments to the templates to increase the number of fragments that are adjacently hybridized; (2) filling in short gaps by trimming any overhanging flaps of any partially hybridized fragments; or (3) filling in short gaps via polymerization. Independent claims are also included for the following: (1) a template-mediated, ligation-oriented method for in vitro non-random shuffling of mutation-containing zones of polynucleotides comprising the steps of the new method above; (2) a template-mediated, ligation-oriented method for in vitro non-random low-homology shuffling of gene families the steps of the new method above; (3) a recombinant polynucleotide obtained by the method; (4) a vector comprising the polynucleotide; (5) a cellular host transformed by the polynucleotide; (6) a protein encoded by the polynucleotide; (7) libraries comprising the recombinant polynucleotide, vector, cellular host or protein; (8) a physical array in which the method can be performed; and (9) logical arrays that stimulate the method or the physical array above; (10) (in vitro) polynucleotide shuffling reaction mixtures comprising: (a) (free) single-stranded fragments of at least two homologous polynucleotides; and (b) at least one devised assembly template upon which at least two of the (restriction) fragments can hybridize adjacently.
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